Oligonucleotide complex compositions and methods of use as gene alteration tools

ABSTRACT

Compositions and methods of treatments of cells are provided for altering the phenotype of a cell by administering an oligonucleotide complex to the cell, the complex having two strands and chemical modifications.

RELATED APPLICATIONS

This application is a national stage application, filed under 35 U.S.C.§371, of International Application No. PCT/US2005/010744, filed on Mar.29, 2005, which claims the benefit of U.S. Ser. No. 60/557,732 filedMar. 29, 2004 and U.S. Ser. No. 60/560,026 filed Apr. 7, 2004, thecontents of which are each herein incorporated by reference in theirentireties.

TECHNICAL FIELD

The present invention relates to the use of specific complexes ofoligonucleotides and their modification products as therapeutic and/orprophylactic agents for the phenotypic and/or genotypic restoration ofmutated genes, genes with inborn errors and/or for switch-on and/orswitch-off of targeted gene(s).

BACKGROUND OF INVENTION

Numerous genetic diseases are caused by mutations in the mammaliangenome. Other sources of genetic diseases are activation of silent genesor the presence of viral genes in the mammalian genome. Several types ofmodifications have been found to be mutated in the genome: deletion ofone or several base pairs, one or several mismatches in the sequence ofthe gene, insertion of one or several bases, or repeat tripletreiteration and absence of a whole or part of a gene.

Genetic diseases caused by mismatches, deletion/insertion of one orseveral base pairs (BP) and repeat triplet mutation in the genes includealbinism, cystic fibrosis, muscular dystrophy and atrophy, sickle cellanemia, hepatic disorders, hemophilia, Crigler-Najjar syndrome, renaltubular acidosis, β-thalassemia, atherosclerosis, Huntington's disease,spinocerebellar ataxia (type 1, 2 and 6), Machado-Joseph disease,myotonic dystrophy, Fragile X (forms A and B) and Frederich's ataxia[Breschel et al., Human Molec. Gen. (1997) 6, 1855-1863; Kmiec, Clin.Invest. (2003) 112, 632-636].

Oligonucleotide complexes and their analogs have been employed as apotential therapeutic for the readout of genes [McManus et al., Nat RevGenet. (2002) 3, 737-747; Nielsen, Curr. Med. Chem. (2001) 8, 545-550;Agrawal et al., Curr. Cancer Drug Targets. (2001) 1, 197-209] and fortargeted gene repair [Kmiec, Clin. Invest. (2003) 112, 632-636].

An approach in the field of gene therapy is introduction ofsequence-specific modification of the genes, using oligonucleotidecomplexes for the phenotypic and/or genotypic restoration of defectivegenes. Approaches to an oligonucleotide-based strategy to achieve thisgoal have been tested. Chimeric RNA/DNA oligonucleotides andsingle-stranded oligonucleotides were developed for site-specificcorrection of episomal and chromosomal target genes [Andersen et al. JMol Med. (2002) 80, 770-81; Alexeev et al., Gene therapy (2002) 9,1667-1675; Kmiec, Clin. Invest. (2003) 112, 632-636; Wu et al., J BiomedSci. (2001) 8, 439-45; Yoon, U.S. Patent Application Publication1999000473872; Davis et al., U.S. Patent Application Publication2000767775; Youn et al., U.S. Patent Application Publication2001000962628; Kmiec et al., U.S. Patent Application Publication2002000260375; Kmiec et al., U.S. Patent Application Publication2002000215432]. Experiments demonstrated the feasibility of usingchimeric RNA/DNA and single stranded oligonucleotides to introduce pointconversions in genes in vitro and in vivo. This gene repair approachrelies on hybridization of the chimera to the target gene, generating amismatch with the targeted point mutation. Restored gene function wasanticipated to occur through activation of endogenous repair systemsthat recognize the created mismatch [Andersen et al., J Mol Med. (2002)80, 770-81; Alexeev et al., Gene therapy (2002) 9, 1667-1675; Kmiec,Clin. Invest. (2003) 112, 632-636; Wu et al., J Biomed Sci. (2001) 8,439-45; Wang et al., (2003) Proc. Natl. Acad. Sci. USA 100,14822-14827]. Double stranded oligonucleotides have been tested for sitespecific gene alteration in plant cells [Arntzen et al., U.S. PatentApplication Publication 1998000129298; Kmiec, U.S. Patent ApplicationPublication 1994000353657].

Triplex forming oligonucleotides also have been employed assequence-specific tools for gene targeting. Triplex formingoligonucleotides bind in the major groove of double stranded DNA, withhigh affinity. Because of this characteristic, triplex formingoligonucleotides have been proposed as tools for the site specificcorrections of targeted genes [Knauert et al., Hum Mol. Genet. (2001)10, 2243-2251; Richardson et al., Drug Target (2002) 10, 133-134; Thounget al., (1993) Angewandte Chemie. Intl. Ed. Eng., 32, 666-690.].

Current targeted gene repair methods are controversial and still at thelevel of development. There is a need for more effective tools, in orderto obtain phenotypic or genotypic restoration of defective genes insomatic tissues.

SUMMARY OF THE INVENTION

The present invention is directed to a method for targeted gene repair.The method is carried out by contacting a non-repaired target RNA withan RNA oligonucleotide complex containing a first oligonucleotide and asecond oligonucleotide, the first oligonucleotide containing a sequencecomplementary to a repaired target RNA, wherein the RNA sequence of thefirst oligonucleotide contains an RNase H-resistant modification, andthe second oligonucleotide contains an RNA sequence complementary to atleast 6 nucleotides of the first oligonucleotide at the site on thesequence of the first oligonucleotide which is not complementary to thenon-repaired target RNA; and hybridizing the complex to the non-repairedtarget RNA in the presence of an RNase, thereby producing a repairedRNA. The first oligonucleotide and the second oligonucleotide areannealed. The repaired target RNA is a wild-type sequence of a gene oranother desired reference sequence. The sequence of non-repaired targetRNA differs from that of the repaired or wild type sequence. Forexample, the non-repaired target RNA is a mutated sequence compared tothe wild type or desired reference sequence. In a preferred embodiment,mutation is a substitution, deletion or insertion of at least one basepair compared to a normal wild type sequence. Prior to contacting anon-repaired target RNA with an RNA oligonucleotide complex thenon-repaired target RNA is contacted with a phosphorothoiate (PS)containing sequence comprising a deoxynucleotide with RNase H resistantflanking ends.

For example, the RNase H-resistant modification is the addition of a2-O-methyl moiety.

The first oligonucleotide is preferably at least 10 nucleotides inlength, e.g. the first oligonucleotide contains about 33 nucleotides.The second oligonucleotide comprises at least 7 nucleotides, the secondoligonucleotide comprises about 11 nucleotides.

The step of, contacting the target RNA preferably occurs within a cell.The cell is in vitro, ex vivo or in vivo. The cell is a mammalian cellsuch as a human cell or an animal cell such as a dog, cat, horse, cow,sheep, or other domesticated or wild animal.

The present invention is directed to a method for treating orameliorating cystic fibrosis in a subject in need thereof, comprisingadministering an RNA oligonucleotide complex directed to a non-repairedtarget RNA, the complex comprising a first oligonucleotide and a secondoligonucleotide, the first oligonucleotide comprising a sequencecomplementary to a repaired target RNA, wherein the RNA sequence of thefirst oligonucleotide comprises an RNase H-resistant modification, andthe second oligonucleotide comprises an RNA sequence complementary to atleast 6 nucleotides of the first oligonucleotide at the site on thesequence of the first oligonucleotide which is not complementary to thenon-repaired target RNA; and wherein administration produces a repairedtargeted RNA, thereby treating or ameliorating cystic fibrosis. Thefirst oligonucleotide and the second oligonucleotide are annealed.

The repaired target RNA is a wild-type sequence. In another embodiment,the non-repaired target RNA is a mutated sequence compared to areference desired sequence. For example a mutated sequence includes asubstitution, deletion or insertion of at least one base pair comparedto a normal wild type sequence.

Prior to administering an RNA oligonucleotide complex, aphosphorothoiate (PS) containing sequence comprising a deoxynucleotidewith RNase H resistant flanking ends is administered. In an example, theRNase H-resistant modification is the addition of a 2-O-methyl moiety.The first oligonucleotide is typically at least 10 nucleotides inlength, e.g. about 33 nucleotides. The second oligonucleotide comprisesat least 7 nucleotides, and an exemplary second oligonucleotidecomprises about 11 nucleotides.

The invention also includes to an RNA oligonucleotide complex formodulating the expression or activity of a cystic fibrosis transmembraneconductance regulator (CFTR) gene product, the complex comprising afirst oligonucleotide and a second oligonucleotide, the firstoligonucleotide comprising the nucleic acid sequence of SEQ ID NO:1 andthe second oligonucleotide comprising the nucleic acid sequence of SEQID NO:2, wherein the first and second oligonucleotide are annealed.

A method for targeted gene repair, is carried out by contacting a targetRNA of a cell with a hybrid DNA/RNA oligonucleotide complex, the complexcomprising a first strand and a second strand, the first strandcomprising a flanking sequence complementary to the target RNA andcentral sequence comprising at least one non-complementary nucleotide ina location opposite a defect of the target RNA, wherein the flankingsequence of the first strand comprises an RNase H-resistant modification3′ and 5′ to the nucleotide, the first strand and the second strandbeing annealed; and hybridizing the complex to the target RNA, wherein arepaired RNA is produced, the RNA comprising a sequence alterationopposite the defect of the target RNA, wherein the genomic target DNAsequence is unaltered. For example, the first strand and second strandare of equal length. Alternatively, the strand is shorter than the firststrand

In another embodiment, the methods further comprise contacting thetarget RNA with a phosphorothoiate (PS) containing sequence of adeoxynucleotide with RNase H resistant flanking ends. Followingcontacting the target RNA with a phosphorothoiate (PS) containingsequence, the target RNA is treated with annealed hybrid oligonucleotidecomplex, and a repaired RNA is produced.

The defect to be repaired is a substitution, deletion, or addition of atleast one base pair compared to a normal wild type sequence. Thealteration is not maintained in a progeny of the cell.

One type of RNase H-resistant modification is the addition of a2-O-methyl moiety. At least one nucleotide of the flanking sequence 3′to the oligonucleotide hybridizable to the target and at least onenucleotide of the flanking sequence 5′ to the oligonucleotidehybridizable to the target comprise the RNase H-resistant modification.The first strand comprises an RNase activating oligonucleotidecomprising a phosphorothioate linkage flanked by RNase H resistantsegments.

Other features and advantages of the invention will be apparent from thefollowing description and from the claims.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 is a set of tracings of whole cell currents of Δ508CFTRexpressing cells. FIG. 1, Top Panel shows representative tracings ofΔ508CFTR expressing cells after treatment with CF4/CF6 were obtainedbefore and after stimulation with a cAMP mixture. The cAMP-activatedcurrents were inhibited by DPC (500 μM). FIG. 1, Bottom Panel, Leftshows that untreated Δ508 cells lacked a cAMP activated whole cellconductance (n=21). FIG. 1, Bottom Panel, Center shows, in contrast,that CF4/CF6 treated Δ508 cells had a robust cAMP response (Linearwhole-cell currents were observed before and after cAMP activation).FIG. 1, Bottom Panel, Right shows that cAMP activation in treated cellswas inhibited by DPC. Similar results were obtained when the initiallyused two-step insertion mechanism was employed.

FIG. 2 is a set of tracings of single channel currents of Δ508CFTRexpressing cells. FIG. 2A, Top Panel, shows a representative tracing ofcurrent observed in Δ508CFTR expressing cells after treatment withCF4/CF6. Currents were obtained in excised inside-out patches. FIG. 2A,Bottom Panel shows that addition of PKA (100 nM) and MgATP (1 mM)induced rapid channel activation. Data are representative of n=12experiments. No activation was observed in control Δ508CFTR expressingcells. FIG. 2B, Top Panel, shows that the PKA-activated Cl⁻ singlechannel currents had a single channel conductance of 12 pS (n=6). FIG.2B, Bottom Panel, the all-point histograms show the details of Cl⁻single channel conductance.

FIG. 3 is a set of bar graphs that show the results of an SPQfluorescence assay. Cl⁻ induced changes in fluorescence were followed incells loaded with the Cl⁻ sensitive dye SPQ. SPQ fluorescence was testedin WT1 cells expressing wild type CFTR, and control and treated Δ508cells in a custom-made chamber, under UV-fluorescence microscopy. AcAMP-induced response (5-15 min) was only observed in WT1 and treatedΔ508 cells (p<0.05). Numbers in parentheses indicate individual cellsanalyzed.

FIG. 4 is a photograph of an agarose gel electrophoretogram showingallele-specific RT-PCR analysis of CFTR mRNA. Samples from WT1 mRNA werediluted into Δ508 mRNA (lanes 2 to 14, from undiluted to 106 order with10-fold serial dilutions) to test efficiency of allele-specific primers(CFFW and CFFM, Table 1). Lanes 2 and 3 show WT1 and Δ508 mRNA amplifiedwith respective primers. The wild type primers recognized WT1 mRNA indilutions of up to 1:10⁵ (Lanes 11 and 12). Gel electrophoresis wasconducted in agarose gels (3%), and a 25 base pair (bp) ladder is shownon the left lane.

FIG. 5 is a photograph of an agarose gel electrophoretogram showingspecific reverse primer analysis of mRNA in samples of RT-PCR materialobtained by amplification with wild type (N1) and mutant (M1) specificreverse primers (Table 1). Lanes 1 and 2 are Δ508 untreated and RT PCRwith N1 and M1 primers respectively. Similarly lanes 3 and 4 are Δ508treated with CF4/CF6 and following RT PCR with N1 and M1 primersrespectively. Gel electrophoresis was performed in 3% agarose gel.

FIG. 6 is an automated tracing by type of nucleotide base obtained fromDNA sequencing of RT-PCR products (SEQ ID NO: 20). Sequence analysis ofthe CF4/CF6 treated Δ508CFTR was conducted by RT-PCR analysis of totalRNA. The PCR materials observed in the gel were subcloned in pCR-Bluntvector and sequenced at the MGH Sequencing Facility. Minor peaksrepresent background. Variations in heights of peaks are due toinstrumental sensitivity

FIG. 7 shows scheme 1. Scheme 1 is a drawing of (A) a hypotheticalscheme for repair of CFTR Δ508 mRNA by CF4/CF6 duplex (SEQ ID NO: 21corresponding to the Wild Type sequence, SEQ ID NO: 22 corresponding tothe Δ508 mRNA sequence, SEQ ID NO: 1 corresponding to the CF4 sequence,and SEQ ID NO: 2 corresponding to the CF6 sequence), and (B) sequencesfound in restored mRNA (SEQ ID NO: 58 corresponding to the N1 sequence,SEQ ID NO: 59 corresponding to the NF1 sequence, SEQ ID NO: 60corresponding to the SNF1 sequence). Bold italic type corresponds tobases of 2′-O-methyl ribosyl oligonucleotides with normalinternucleoside phosphate bonds (CF4 oligonucleotide). Regular typecorresponds to natural RNA. Bold plus underlined type corresponds toinserted bases of ribonucleotides. Shaded bases in the CF4 and Δ508 mRNAindicate non-complementary Watson-Crick base pairs. Sequence analysis ofRT-PCR products obtained by using different primers is also shown. (a)

in square indicates that in 7 out of 10 sequencings this A was present,but in 3 other sequencings was absent, leaving 3 deletions. (b) Dashesindicate deletions, possibly induced by PCR. (c) There are no deletions.

in square indicates that in this position U sometimes is replaced byanother nucleotide.

FIG. 8 shows scheme 2. Scheme 2 is a drawing of a hypotheticalsite-specific deletion/insertion mechanism in the RNA containingmismatch region, with the two-step nucleotide complex technique. Redcorresponds to targeted nucleotide(s), black and white correspond tohybrid oligonucleotide for RNase-H activation, yellow corresponds tonatural RNA, grey corresponds to template oligonucleotide ininsertion/deletion duplex, and blue corresponds to inserted/repairedsegment of RNA

FIG. 9 shows scheme 3. Scheme 3 is a drawing of a hypotheticalsite-specific deletion/insertion mechanism in the RNA containingmismatch region, with the one step oligonucleotide complex technique.Red corresponds to targeted nucleotide(s), yellow corresponds to naturalRNA, grey corresponds to template oligonucleotide in insertion/deletionduplex, and blue corresponds to inserted/repaired segment of RNA

FIG. 10 shows scheme 4. Scheme 4 is a drawing of a hypotheticalsite-specific deletion/insertion mechanism in the RNA containing deletedregion, with the two-step oligonucleotide complex technique. Black andwhite correspond to hybrid oligonucleotide for RNase-H activation,yellow corresponds to natural RNA, grey corresponds to templateoligonucleotide in insertion/deletion duplex, and blue corresponds toinserted/repaired segment of RNA

FIG. 11 shows scheme 5. Scheme 5 is a drawing of a hypotheticalsite-specific insertion/deletion mechanism in the RNA containing deletedregion, with the one step oligonucleotide complex technique. Yellowcorresponds to natural RNA, grey corresponds to template oligonucleotidein “insertion duplex”, and blue corresponds to inserted/repaired segmentof RNA (or DNA).

FIG. 12 shows scheme 6. Scheme 6 is a drawing of a hypotheticalsite-specific deletion/insertion mechanism in the RNA containing repeatmutations, with the two-step oligonucleotide complex technique. Redcorresponds to targeted nucleotide(s), black and white correspond tohybrid oligonucleotide for RNase-H activation, yellow corresponds tonatural RNA, grey corresponds to template oligonucleotide ininsertion/deletion duplex, and blue corresponds to inserted/repairedsegment of RNA

FIG. 13 shows scheme 7. Scheme 7 is a drawing of a hypotheticalsite-specific deletion/insertion mechanism in the RNA containing repeatmutations, with the one step oligonucleotide complex technique. Redcorresponds to targeted nucleotide(s), yellow corresponds to naturalRNA, grey corresponds to template oligonucleotide in insertion/deletionduplex, and blue corresponds to inserted/repaired segment of RNA. TheXXX indicates the size of the base of the oligonucleotide loop. It mayvary between zero and twenty.

FIG. 14 shows scheme 8. Scheme 8 is a drawing of a hypotheticalsite-specific frame changing mechanism in the active RNA, with thetwo-step oligonucleotide complex technique. Red corresponds to targetednucleotide(s), black and white correspond to hybrid oligonucleotide forRNase-H activation, yellow corresponds to natural RNA, grey correspondsto template oligonucleotide in insertion/deletion duplex, and bluecorresponds to inserted/frame changed segment of RNA

FIG. 15 shows scheme 9. Scheme 9 is a drawing of a hypotheticalsite-specific frame changing mechanism in the active RNA, with the onestep oligonucleotide complex technique. Red corresponds to targetednucleotide(s), yellow corresponds to natural RNA, grey corresponds totemplate oligonucleotide in insertion/deletion duplex, and bluecorresponds to inserted/frame changed segment of RNA.

FIG. 16 shows scheme 10. Scheme 10 is a drawing of a hypotheticalsite-specific transcription or translation start site insertionmechanism in the active RNA, with the two-step oligonucleotide complextechnique. Red corresponds to targeted nucleotide(s), black and whitecorrespond to hybrid oligonucleotide for RNase-H activation, yellowcorresponds to natural RNA, grey corresponds to template oligonucleotidein insertion/deletion duplex, and blue corresponds to inserted/framechanged segment of RNA.

FIG. 17 shows scheme 11. Scheme 11 is a drawing of a hypothetical schemefor repair of Inactive tyrosinase by two-step oligonucleotide complextechnique (depicting SEQ ID NO: 23, SEQ ID NO: 23, SEQ ID NO: 24, SEQ IDNO: 25 (left side schematic sequence), SEQ ID NO: 26 (right sideschematic sequence), SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ IDNO: 30, and SEQ ID NO: 31, from top to bottom, respectively). Bolditalic type corresponds to natural RNA. Small caps correspond to PS ODN.Shaded corresponds 2′-o-methylribosyl oligonucleotides, withinternucleoside phosphate bonds. Underline base corresponds targetedbase. Base in squire correspond replaced base.

FIG. 18 shows scheme 12. Scheme 12 is a drawing of a hypothetical schemefor repair of Inactive tyrosinase by one-step oligonucleotide complextechnique (depicting SEQ ID NO: 32, SEQ ID NO: 32, SEQ ID NO: 33, SEQ IDNO: 34, and SEQ ID NO: 35, from top to bottom, respectively). Bolditalic type corresponds to natural RNA. Small caps correspond tothiophosphate oligodeoxynucleotide. Shaded corresponds2′-o-methylribosyl oligonucleotides, with internucleoside phosphatebonds. Underline base corresponds targeted base. Base in squirecorrespond replaced base.

FIG. 19 shows scheme 13. Scheme 13 is a drawing of a hypothetical schemefor repair of Cystic Fibrosis Δ508 mRNA by two-step oligonucleotidecomplex technique (depicting SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:22, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, and SEQ ID NO: 40, from top to bottom, respectively). Bolditalic type corresponds to natural RNA. Small caps correspond tothiophosphate oligodeoxynucleotide. Shaded corresponds2′-o-methylribosyl oligonucleotides, with internucleoside phosphatebonds. * and _(— — —) corresponds deletion region of mutated mRNA.

FIG. 20 shows scheme 14. Scheme 14 is a drawing of a hypothetical schemefor repair of Cystic Fibrosis Δ508 mRNA by one step oligonucleotidecomplex technique (depicting SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:22, SEQ ID NO: 22, SEQ ID NO: 41, SEQ ID NO: 39, and SEQ ID NO: 42, fromtop to bottom, respectively). Bold italic type corresponds to naturalRNA. Shaded corresponds 2′-o-methylribosyl oligonucleotides, withinternucleoside phosphate bonds. * and _(— — —) correspond to a deletionregion of mutated mRNA.

FIG. 21 shows scheme 15. Scheme 15 shows the insertion into the gene forHuntingtin protein with HT3 followed by HT4/HT6 duplex oligonucleotides(depicting SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 43, SEQ ID NO: 14,SEQ ID NO: 45 (left of cut mRNA), SEQ ID NO: 46 (right of cut mRNA), SEQID NO: 15, SEQ ID NO: 16, SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO:49, from top to bottom, respectively). Shaded type in sequencescorrespond to bases of 2′-O-methylribosyl oligonucleotides, withinternucleoside phosphate bonds. Regular type corresponds to naturalRNA. Bold type in square corresponds to the inserted segment of mRNA.There is purposely deleted one base (x) in modified mRNA. Low case pcorresponds to terminal phosphate groups.

FIG. 22 shows scheme 16. Scheme 16 shows the insertion into the Aim-1gene with A1b3 followed by A1b4/A1b6 duplex oligonucleotides (depictingSEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO:54, SEQ ID NO: 17, SEQ ID NO: 55 (left of cut mRNA), SEQ ID NO: 56(right of cut mRNA), SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, andSEQ ID NO: 57, from top to bottom, respectively). Shaded type insequences corresponds to bases of 2′-o-methylribosyl oligonucleotides,with internucleoside phosphate bonds. Regular type corresponds tonatural RNA. Strikethrough in the sequence of repaired mRNA correspondsto inserted segment of mRNA. Low case p corresponds to terminalphosphate groups.

DETAILED DESCRIPTION

The present invention is directed to a method for targeted gene repair,comprising contacting a non-repaired target RNA with an RNAoligonucleotide complex comprising a first oligonucleotide and a secondoligonucleotide, the first oligonucleotide comprising a sequencecomplementary to a repaired target RNA, wherein the RNA sequence of thefirst oligonucleotide comprises an RNase H-resistant modification, andthe second oligonucleotide comprises an RNA sequence complementary to atleast 6 nucleotides of the first oligonucleotide at the site on thesequence of the first oligonucleotide which is not complementary to thenon-repaired target RNA; and hybridizing the complex to the non-repairedtarget RNA in the presence of an RNase, wherein a repaired RNA isproduced. Preferably, any genetic defect, e.g., a substitution,deletion, or addition of at least one basepair, compared to a normalwild type sequence, is phenotypically changed.

The first oligonucleotide and the second oligonucleotide are annealed.The repaired target RNA is an optionally wild-type sequence, and thenon-repaired target RNA is a mutated sequence. The mutation is asubstitution, deletion or insertion of at least one base pair comparedto a normal wild type sequence. In one embodiment, prior to contacting anon-repaired target RNA with an RNA oligonucleotide complex thenon-repaired target RNA is contacted with a phosphorothoiate (PS)containing sequence comprising a deoxynucleotide with RNase H resistantflanking ends. The RNase H-resistant modification is the addition of a2-O-methyl moiety. The first oligonucleotide is at least 10 nucleotidesin length. In a preferred embodiment, the first oligonucleotidecomprises about 33 nucleotides. In another embodiment, the secondoligonucleotide comprises at least 7 nucleotides. In a preferredembodiment, the second oligonucleotide comprises about 11 nucleotides.Contacting the target RNA occurs within a cell. In preferred embodiment,cell is in vitro, ex vivo or in vivo. In another preferred embodiment,the cell is a mammalian cell. In a more preferred embodiment, the cellis a human cell.

The present invention is also directed to a method for treating orameliorating cystic fibrosis in a subject in need thereof, comprisingadministering an RNA oligonucleotide complex directed to a non-repairedtarget RNA, the complex comprising a first oligonucleotide and a secondoligonucleotide, the first oligonucleotide comprising a sequencecomplementary to a repaired target RNA, wherein the RNA sequence of thefirst oligonucleotide comprises an RNase H-resistant modification, andthe second oligonucleotide comprises an RNA sequence complementary to atleast 6 nucleotides of the first oligonucleotide at the site on thesequence of the first oligonucleotide which is not complementary to thenon-repaired target RNA; and wherein administration produces a repairedtargeted RNA, thereby treating or ameliorating cystic fibrosis.

The first oligonucleotide and the second oligonucleotide are annealed.

The repaired target RNA is a wild-type sequence. In another embodiment,the non-repaired target RNA is a mutated sequence. In a preferredembodiment, mutation is a substitution, deletion or insertion of atleast one base pair compared to a normal wild type sequence.

Prior to administering an RNA oligonucleotide complex, aphosphorothoiate (PS) containing sequence comprising a deoxynucleotidewith RNase H resistant flanking ends is administered. RNase H-resistantmodification is the addition of a 2-O-methyl moiety. The firstoligonucleotide is at least 10 nucleotides in length. In a preferredembodiment, the first oligonucleotide comprises about 33 nucleotides. Inanother embodiment, the second oligonucleotide comprises at least 7nucleotides. In a preferred embodiment, the second oligonucleotidecomprises about 11 nucleotides.

The present invention is also directed to an RNA oligonucleotide complexfor modulating the expression or activity of a disease-associate geneproduct cystic fibrosis transmembrane conductance regulator (CFTR) geneproduct, the complex comprising a first oligonucleotide and a secondoligonucleotide, the first oligonucleotide comprising the nucleic acidsequence of SEQ ID NO:1 and the second oligonucleotide comprising thenucleic acid sequence of SEQ ID NO:2, wherein the first and secondoligonucleotide are annealed.

The present invention includes method for targeted gene repair,comprising contacting a target RNA of a cell with a hybrid DNA/RNAoligonucleotide complex, the complex comprising a first strand and asecond strand, the first strand comprising a flanking sequencecomplementary to the target RNA and central sequence comprising at leastone non-complementary nucleotide in a location opposite a defect of thetarget RNA, wherein the flanking sequence of the first strand comprisesan RNase H-resistant modification 3′ and 5′ to the nucleotide, the firststrand and the second strand being annealed; and hybridizing the complexto the target RNA, wherein a repaired RNA is produced, the RNAcomprising a sequence alteration opposite the defect of the target RNA,wherein the genomic target DNA sequence is unaltered.

The complex contains a first strand and a second strand. The first andsecond strand is annealed to one another to form the complex. The firststrand contains a flanking sequence that is complementary to sequence ofthe target RNA (“hybridized ends”) and a central sequence (locatedbetween the flanking sequence) that contains at least onenon-complementary nucleotide or deletion in a location opposite a defectof the target RNA. The nucleotide sequence of the first strand iscomplementary to the sequence of a normal wild type gene. The centralsequence, which is RNase-sensitive, is at least a triplet, and ispreferably at least 4, 5, 6, or more nucleotides in length. The flankingsequence of the first strand includes an RNase H-resistant modification,e.g., addition of a 2-O-methyl moiety, or other modifications, to both a3′ and 5′ to the nucleotide hybridizable to the target sequence of thegene.

The second strand of the complex is of the same length as the firststrand, or is shorter than first strand of the complex, and the secondand first strands are complementary to and annealed to one another. Thecomplex is allowed to hybridize to target genomic RNA of the targetcell, and a repaired RNA is produced in the cell. The repaired RNAcontains a sequence alteration opposite the defect of the target RNA,and the genomic target DNA sequence remains unaltered. The RNAalteration or phenotypic change accomplished by the method is notmaintained in a progeny of the cell, i.e., is not heritable.

The flanking sequence of the first strand contains at least fourcomplementary nucleotides (relative to the genomic target RNA) 3′ to thehybridizable sequence of the targeted gene (of the target RNA and firststrand RNA oligonucletide) and at least four complementary nucleotides(relative to the genomic target RNA) 5′ to the hybridizable sequence ofthe targeted gene. The flanking portion of the complex sequence containsan RNase H-resistant modification, whereas the central portion of thesequence (opposite the defect) does not contain such a modification andis therefore nuclease and RNase sensitive, i.e., the nucleotide bonds inthe central region are enzymatically cleaved.

The first strand is at least 10 nucleotides in length. For example, thefirst strand contains at least about 10, 12, 14, 15, 16, 18, 20, 30, 40,or 50 or more nucleotides, the flanking sequence of which contains atleast 2 nucleotides that are complementary to target RNA in the regionof the defect. The complementary flanking sequence on either side of thedefect is about 5, 10, 15, 20, 25 or more nucleotides in length. Thesecond strand of the oligonucleotide complex is at least about 7nucleotides in length and is optionally longer, e.g., 9, 11, 12, 15, or20 nucleotides.

Another approach involves hybridization of a PS containing segment of adeoxyribonucleotide with RNase H resistant flanking ends, resulting inan RNase-catalized deletion of a small central segment of mRNA. Thisoligonucleotide is then washed out of the incubation medium and isreplaced by a double-stranded, annealed hybrid oligonucleotide. Thedouble-stranded, annealed hybrid oligonucleotide hybridizes with thetargeted mRNA, and inserts an oligonucleotide segment complementary tothe hybridizable oligonucleotide part of the duplex repair complex.

Nucleotide modifications are selected from the group 2′-O-methylribosyl; phosphorothioate; peptide nucleic acid; 2′-halo, 2′-fluoro,2′-alkyl, 2′-alkoxylalkyl, bridged sugars and similar chemical moieties;methylphosphonate, ethylphosphonate, phosphoramidate, thiophosphate,dithiophosphate, morpholino, boronophosphate, morpholinophosphateinternucleoside linkages and similar chemical moieties; derivatizedheterocycles, and sugars.

The method is utilized in analytical research applications as well as inpreventative, diagnostic and therapeutic applications. Complexes areadministered to a subject, e.g., a human patient, suffering from adisorder associated with a genetic defect, or to a cell obtained from apatient having a genetic disease. Cells or tissue are contacted invitro, in vivo or ex vivo. In some embodiments, the cell is an isolatedcell in a culture. The method can further involve isolating andsequencing RNA from a contacted cell having a different phenotype fromcells from the patient or the culture, wherein altering expression ofthe target gene is altering an RNA nucleotide sequence.

A target gene such as one associated with a disease state is diagnosedby detecting a mutation compared to the sequence of a normal wild typegene. The central segment of the oligonucleotide repair complex has anormal sequence compared to the target gene. A modification confersribonuclease resistance. Alternatively, a modification confers inductionof ribonuclease activity in the cell. At least one nucleotide of thecomplex is a 2′-O-methyl ribosyl nucleotide. For example, there is aplurality of 2′-O-methyl ribosyl nucleotides. The second strand canfurther contain a small chemical group at a 5′ or 3′ terminus, or thesecond strand further contains a small chemical group at both 5′ and 3′termini. The modification can be located at a nucleotide in a 3′ or a 5′terminus, or both. The second strand further comprises a modificationcomprising a small chemical group in a 3′ or a 5′ terminus. For example,the small chemical group is selected from phosphate, diphosphate,triphosphate, thiophosphate, dithiophosphate, aldehyde, carboxy,dihydroxy, hydroxyl, methyl, ethyl, sulfhydryl, sulfate, and boronate.The central segment comprises at least one modification, which is aphosphorothioate.

The genetic disease is selected from the group of albinism, cysticfibrosis, muscular dystrophy, myotonic dystrophy, muscular atrophy,sickle cell anemia, hepatic disorder, hemophilia, Crigler-Najjarsyndrome, renal tubular acidosis, β-thalassemia, atherosclerosis,Huntington's disease, spinocerebellar ataxia, Machado-Joseph disease,Fragile X, Frederich's ataxia, adenosine deaminase deficiency,hepatocellular carcinoma, hepatoblastoma, osteosarcoma, adenocarcinoma,breast cancer, adrenocortical carcinoma, non-Hodgkin lymphoma,glioblastoma, rhabdomyosarcoma, glioma, sickle cell anemia, haemolyticanaemia, retinoblastoma, ovarian cancer, cystic fibrosis, congentialabsence of vas deferens, melanoma, adenomatous polyposis, colorectalcancer, factor V deficiency, thrombosis, haemophilia A, haemophilia B,warfarin sensitivity, thalassaemia alpha, haemoglobin variant,haemoglobin H disease, non-polyposis colorectal cancer, endometrialcancer, early onset colorectal cancer, apolipoprotein E,hypercholesterolaemia, Gilbert syndrome, Alzheimer's disease andSchizophrenia.

The methods and compositions of the present invention are used to treator ameliorate cystic fibrosis, Huntington's disease or albinism. Morespecifically, methods and compositions of the present invention are usedto treat or ameliorate at least one symptom of cystic fibrosis,Huntington's disease or albinism.

Cystic fibrosis can be diagnosed at birth, but most often is diagnosedduring the early childhood years in young children (by the age of 3years) who have had a history of respiratory infections, excessive fatin their stools, and who have poor weight gain. Nearly 8 percent ofpeople with cystic fibrosis are diagnosed at 18 years of age or olderbecause they have experienced only mild symptoms of cystic fibrosis.Because one of cystic fibrosis's major symptoms is respiratoryinfection, a cystic fibrosis diagnosis sometimes may be confused withother respiratory conditions such as asthma, pneumonia, or chronicbronchitis.

The methods and compositions of the present invention limit, reduce,lessen or eliminate one or more symptoms of cystic fibrosis. Symptoms ofcystic fibrosis include but are not limited to: thick, viscous mucussecretions in the lungs; repeated infections: The accumulation ofsticky, thick mucus in the lungs creates a favorable environment forinfectious microorganisms to inhabit and flourish; stools, pale or claycolored, foul smelling, or stools that float; recurrent pneumonia;chronic cough, possibly with blood streaking; wheezing; bronchitis;chronic sinusitis; asthma; nasal polyps (fleshy growths inside thenose); weight loss, failure to thrive in infants, abdominal swelling;excessive salt in sweat, dehydration; failure of newborn to pass stool;abdominal pain, flatulence; fatigue; enlarged fingertips (clubbing);changes in color and amount of sputum (material coughed up from thelungs)

Conditions such as late onset of puberty, intestinal obstruction,inflammation of the pancreas, cirrhosis (a liver condition), andinfertility may also be signs of cystic fibrosis. Cystic fibrosissymptoms does not follow the same pattern in all patients but affectsdifferent people in different ways and to varying degrees. However, thebasic problem is the same—an abnormality in the glands, which produce orsecrete sweat and mucus. Sweat cools the body; mucus lubricates therespiratory, digestive, and reproductive systems, and prevents tissuesfrom drying out, protecting them from infection.

People with Cystic fibrosis lose excessive amounts of salt when theysweat. This can upset the balance of minerals in the blood, which maycause abnormal heart rhythms. Going into shock is also a risk.

Mucus in Cystic fibrosis patients is very thick and accumulates in theintestines and lungs. The result is malnutrition, poor growth, frequentrespiratory infections, breathing difficulties, and eventually permanentlung damage. Lung disease is the usual cause of death in most patients.(Loss of lung function is a major medical problem in most patients withcystic fibrosis. The average person with cystic fibrosis experiences agradual worsening of lung function each year due to infection andinflammation. In people with cystic fibrosis, loss of lung functionprimarily is caused by blockage of air passages with infected mucus. Thethick mucus plugs the air passages of the lungs and must be broken upand removed. The repeated lung infections also can cause permanentscarring of the lungs. Many adults with cystic fibrosis also developsymptoms of chronic sinus infections.)

Cystic fibrosis can cause various other medical problems. These symptomsmay include sinusitis (inflammation of the nasal sinuses, which arecavities in the skull behind, above, and on both sides of the nose),nasal polyps (fleshy growths inside the nose), clubbing (rounding andenlargement of fingers and toes), pneumothorax (rupture of lung tissueand trapping of air between the lung and the chest wall), hemoptysis(coughing of blood), cor pulmonale (enlargement of the right side of theheart), abdominal pain and discomfort, gassiness (too much gas in theintestine), and rectal prolapse (protrusion of the rectum through theanus). Liver disease, diabetes, inflammation of the pancreas andgallstones also occur in some people with Cystic fibrosis. (The pancreassecretes enzymes into the intestines to aid in the digestion of foodsand liquids. However, patients with cystic fibrosis who suffer frompancreatic insufficiency do not secrete enough enzymes to derive propernutritional benefit from their food. Also, the intestines are lined withthick, sticky mucus that prevents any secreted enzymes from reaching thefood to be digested. Due to the inability to properly digest food, aperson with cystic fibrosis produces large, greasy stools, which arelighter in color than normal and have a very foul odor. Patients alsomay suffer from abdominal cramping. To treat this problem, patients aregiven supplemental pancreatic enzymes that help them digest their food.

The pancreas also secretes insulin to process sugars. Some people withcystic fibrosis develop a specific type of diabetes called CysticFibrosis-Related Diabetes. Cystic fibrosis also can result in damage tothe liver. In some patients with cystic fibrosis, the bile ducts, whichtransport bile from the liver to the intestines, become blocked. Thiscondition can result in irreversible liver damage).

Huntington's disease is caused by a faulty gene and is diagnosed bydetecting defective gene sequence.

Most genes in the body are present in two copies, one from the motherand one from the father. The gene (which is called “Huntington”) thatcauses Huntington's disease is dominant. This means that if a personinherits one copy of this gene from either parent, they will go on todevelop the disease at some point in their life. A person who has oneparent with Huntington's disease has a 50% chance of inheriting thefaulty gene. This risk is 50% for each child and is not altered bygender or whether brothers and sisters are affected. Only people whohave the faulty gene are capable of passing it on to their own children.Occasionally some people can develop the condition even though there isno family history of it. This is usually because previous generationswere not diagnosed as a result of early death from other causes, or lossof contact through adoption.

Huntington's disease causes damage to brain cells in the areas of thebrain involved in the control of movement, planning and motivation.People usually have symptoms for up to 10 years before they find outthey have Huntington's disease. Most people are diagnosed between theages of 30 and 50, although this can happen much earlier or later.Symptoms are often overlooked, as they are mild and commonly experiencedby well people.

The methods and compositions of the present invention limit, reduce,lessen or eliminate one or more symptoms of Huntington's disease.Symptoms of Huntington's disease include but are not limited to: mildtremor, clumsiness, lack of concentration, difficulty rememberingthings, mood changes, including depression, sometimes, aggressiveantisocial behavior.

As the disease continues, the symptoms become progressively worse, andlead to incapacitation. Physical symptoms include chorea (involuntarymovements of the limbs, face and body). Chorea may lead to difficultywalking, speaking and swallowing. Choking is a particular hazard, due toreduced ability to control the muscles of the tongue, neck anddiaphragm. People often lose weight because they have difficulty eatingand burn more calories due to the continuous movement.

Emotional symptoms include depression, not only because of the burden ofhaving a progressive disorder, but also as a direct result of the damageto certain brain cells. People often become frustrated at being unableto work or carry out previously simple tasks. They also seem to behavestubbornly, probably due to a reduced ability to react flexibly and tounderstand the needs and emotions of others. People with Huntington'smay also become more irritable and antisocial than usual, or makeinappropriate sexual advances.

Cognitive symptoms include a loss of drive and initiative. People withHuntington's may appear to be lazy or uninterested in life, spendingdays doing little or neglecting personal hygiene. They may also lose theability to organise themselves, as planning skills and ability to carryout more than one task at once deteriorate. In later stages, people mayget memory loss and be less able to understand speech. The behavioralchanges that occur in people with Huntington's disease are often themost distressing for them, their family and their carriers. People withpreviously full and active lives gradually lose their independence andmobility. At the same time, their personality may become gradually moreself-centered and apathetic, straining personal relationships.

A juvenile form of Huntington's disease also exists. Symptoms startbefore the age of 21 and the condition is usually inherited from thefather. Symptoms are similar to those of adult Huntington's, but developmore rapidly and include muscular rigidity and fits.

The methods and compositions of the present invention limit, reduce,lessen or eliminate one or more symptoms of albinism. Albinism includesHypopigmentation, oculocutaneous albinism and ocular albinism. Symptomsof nalbinism include but are not limited to: absence of pigment from thehair, skin, or iris of eyes; patchy absence of pigment (skin color,patchy); lighter than normal skin and hair, complete albinism; rapid eyemovements (nystagmus); strabismus (eyes not tracking properly);photophobia (avoidance of light because of discomfort); decreased visualacuity and functional blindness. The disorders diagnosed by detection ofphysical symptoms or detection of the underlying genetic defects.

The oligonucleotide complex technique provided herein is a novelapproach to modulation of genetic diseases, including but not limited tothose diseases mentioned above. The oligonucleotide complex techniqueprovides functional restoration of the mutated genes bydeletion/insertion of a segment of RNA (or DNA). The oligonucleotidecomplex technique method inserts one or more deleted nucleotide(s) intothe targeted region of specific RNA (or DNA). The oligonucleotidecomplex technique facilitates simultaneous correction of more then onemutation with a single set of oligonucleotide complex into a singletargeted RNA (or DNA) gene.

Site-specific insertion of the specific RNA (or DNA) genes with theoligonucleotide complex technique may require one or more treatmentsteps with the oligonucleotide complex. Site-specific alteration of genewith the oligonucleotide complex technique may be performed in one ormore targeted genes by using several oligonucleotide complex setsconsecutively as required, or even simultaneously. For enhancingtargeted gene repair efficiency using the oligonucleotide complextechnique this complex is used synergistically with other known orpotential therapeutic compounds. The oligonucleotide complex techniqueis used for targeting one or several harmful genes one by one orsimultaneously. The oligonucleotide complex technique is used forextension of translation of targeted genes (by elimination ofundesirable stop codons). The oligonucleotide complex technique is usedfor creation of “new genes” by insertion of transcription or translationsites in certain regions of the mammalian genome

The oligonucleotide complex technique is used for deletion or insertionof purine or pyrimidine repeats at the ends of different genomic RNA orDNAs. The oligonucleotide complex technique is used for the deletion ordiminution of a region of RNA containing a reiteration of excesstriplets. The oligonucleotide complex technique provides insertion ofUGU in a sufficient fraction of Δ508 mRNA to induce phenotypic reversionin a tissue culture cell line. The oligonucleotide complex techniqueaccording of SPQ data indicates suppression of the chloride anionconductance, or in the other words phenotypic restoration of a mutatedΔ508 gene. The oligonucleotide complex technique according to subcloningdata presented herein shows approximately 30 percent insertion of UGU inthe site of the Δ508 mRNA UUU triplet deletion. The oligonucleotidecomplex technique is used for deletion or insertion of purine orpyrimidine repeats at the ends of specific genomic RNA or DNAs. Theoligonucleotide complex technique inserts UGU in the site of the Δ508mRNA UUU triplet deletion with one step addition of the oligonucleotidedeletion/insertion hybridized RNA/modified RNA duplex. Theoligonucleotide complex technique inserts UGU in the site of the Δ508mRNA UUU triplet deletion with more than one (two) step treatment. Thefirst step treatment was performed with RNase H (or otherendonucleolytic enzyme) deleting single strand RNA. The second step ofthe treatment was performed by the oligonucleotide deletion/insertionRNA/modified RNA duplex. The oligonucleotide complex technique mayrequire more then one step treatment (i.e. consecutive steps) for therestoration of targeted RNA (DNA) gene(s). RNase H (or otherendonucleolytic enzyme) deletes a specific region of RNA, plus thepre-hybridized duplex of oligonucleotides involved in theoligonucleotide complex technique include, but are not limited to,standard oligonucleotides, modified oligonucleotides, standard andmodified oligonucleotides with different sequential alteration, standardor modified oligonucleotide in the middle and flanked by differentderivatives of oligonucleotides, derivatives of oligonucleotidesfacilitating different endonucleolytic deletions and/or some other “geneinsertion/deletion” catalysis, oligonucleotides conjugated withdifferent chemical groups (such as, but not limited to, intercalators,groove binders, alkylating reagents, photoactive groups (such aspsoraren) and other moieties).

Compositions of oligonucleotide derivatives in the oligonucleotidecomplex technique include, but are not limited to standard and modifiedoligonucleotides with different sequential alteration. Oligonucleotidederivatives in the oligonucleotide complex technique may contain smallchemical groups at the 5′- and/or 3′-end, including but not limited to,phosphate, diphosphate, triphosphate, thiophosphate, dithiophosphate,aldehyde, carboxyl, dihydroxyl, hydroxyl, methyl, ethyl, sulfhydryl,sulfate, Boronates, and similar chemical moieties. Oligonucleotidederivatives in the oligonucleotide complex technique may include, butare not limited to PNA; 2′-halo, 2′-fluoro, 2′-alkil, 2′-alkoxylalkyl,bridged sugars and similar chemical moieties; methylphosphonate,ethylphosphonate, phosphoramidate, thiophosphate, dithiophosphate,morpholino, Boronophosphate, morpholinophosphate internucleosidelinkages and similar chemical moieties; oligonucleotides withderivatized heterocycles, sugars and/or internucleoside linkages andsimilar chemical moieties. Oligonucleotide derivatives in theoligonucleotide complex technique may include, but are not limited onlyto derivatized heterocycles, sugars or internucleoside linkages, butalso to combination of these moieties mentioned above.

RNase H- (or other endonucleolytic enzyme-) activating single strandedoligonucleotide and pre-hybridized duplex of oligonucleotides involvedin the oligonucleotide complex technique method may be four or more (upto three hundred) base long. The deletion/insertion oligonucleotideduplex used in the oligonucleotide complex technique method maybecomposed of oligonucleotides of different length. The deletion/insertionoligonucleotide duplex employed in the oligonucleotide complex techniquemay possess the 5′- and/or 3′-“sticky ends”. The length of “the stickyends” in the deletion/insertion oligonucleotide duplex employed in theoligonucleotide complex technique may be one base and more (up to 40-60bases). The double strand part of the deletion/insertion oligonucleotideduplex employed in the oligonucleotide complex technique may be 4-5 basepairs or more (up to 100 base pairs).

Sources of the derivatives of oligonucleotide used in theoligonucleotide complex technique method are chemically or biologicallysynthesized. The oligonucleotide complex technique is also useful totarget with an oligonucleotide sequence specifically the deletion orinsertion of a portion of a gene from a pathogenic bacterium, virus,insect, arthropod, parasite, land or marine plant or other livingorganism; to silence a virulence factor, thereby rendering that organisminto a non-pathogenic one. This technique is used for preparation of avaccine against the pathogen, or a therapeutic treatment by spray orother means of obtaining entry to the pathogen.

The oligonucleotide complex technique is used for thesuppression/inactivation of biological pathogens in body fluids eitherin vitro, in vivo or ex vivo. The oligonucleotide complex technique isused for therapeutic purposes, diagnostic analytical purposes or as atool for a laboratory research.

Cystic Fibrosis

Cystic Fibrosis (CF) is a lethal disorder caused by mutations in theCFTR gene encoding the CFTR channel [Riordan, et al., J. R., (1989)Science, 245, 1066-1073; Kerem et al., (1989) Science, 245, 1073-1080];Burke (2003), Eng. J. Med. 349, 969-974]. CFTR, a cAMP-activated anionchannel [Anderson et al., (1991) Science, 251, 679-682; Bear et al.,(1992) Cell, 68, 809-818], is associated with the dysfunctionality ofepithelia in several tissues [Crawford et al., (1991) Proc. Natl. Acad.Sci. USA 88, 9262-9266] including lungs, pancreas, intestine, sweatglands and kidneys. CF is the most common lethal genetic disease ofCaucasians, affecting 1 in 2000 individuals. More than 150 mutationshave been identified in the CFTR gene [Tsui, (1992) Trends Genet. 8,391-398], associated with a pleiotropic spectrum of CF phenotypes[Cutting, (1993), J. Bioenerg. Biomembr. 25, 7-10; Strandvik et al.,(2001), Genet. Test. 5, 235-242]. New mutations have been found withdistinctive impacts on CF populations [Gilfillan et al., (1998), J. Med.Genet. 35, 122-125; Onay et al., (1998), Hum. Genet. 102, 224-230;Visich et al., (2002), Clin. Genet. 61, 207-213]. However, the mostfrequent CFTR mutation, accounting for almost 75% of all cases of thedisease, is a deletion of three bases (TTT), encoding the amino acidphenylalanine in position 508 of the translation sequence, accompaniedby C to U replacement immediately 5′ to the deletion. This deletion isoften accompanied by a mismatch immediately adjacent in the 5′ directionto the deletion: in a U in place of a C.

The Δ508 CFTR is a misfolded but partially functional channel protein[Li et al., (1993) Nature Genet. 3, 311-316; Pasyk et al., (1995) J.Biol. Chem. 270, 12347-12350], unable to translocate perfectly to targetplasma membranes [Denning et al., (1992) Nature, 358, 761-764; Cheng etal., (1990), Cell, 63, 827-834]. Several chemical and pharmacologicalstrategies have been attempted, to rescue the CF phenotype at thecellular level, to bring Δ508 CFTR to the plasma membrane. Partialsuccess has also been achieved by adenoviral vector infection [Boucheret al., (1994), Hum. Gene Ther. 5, 615-639; Teramoto et al., (1998), J.Virol. 72, 8904-8912] and re-insertion of the wild type CFTR gene into aCF genetic background. More recently, a novel repair strategy was used,based on trans-splicing of the Δ508CFTR pre-mRNA [Liu et al., (2002),Biotechnol. 20, 47-52; Puttaraju et al., (2001), Mol. Ther. 4, 105-114;Mansfield et al., (2000) Gene Ther. 7, 1885-1895]. Gentamycin has alsobeen found to induce a correction of faulty CFTR function in CF, causedby CFTR stop mutations [Bedwell et al., (1997) Nature Med., 3,1280-1284], rather than by the TTT deletion.

Loss or suppression of disease-associated gene function by antisenseoligonucleotide technologies involves specific inhibition of DNA, RNAand protein expression [Zamecnik et al., (1978), Proc. Natl. Acad. Sci.USA 74, 280-284; Stephenson et al., (1978) Proc. Natl. Acad. Sci. USA75, 285-288]. This is based in good part on complementary hybridizationof synthetic oligonucleotides with the natural sequences in either DNAor RNA [Temsarnani et al., (1994) Antisense Res. Devel. 4, 279-284].Triplex-forming oligonucleotides have also been used for themodification of cellular gene function [Felsenfeld et al., (1957) J. Am.Chem. Soc., 79, 2023-2024; Thoung et al., (1993) Angewandte Chemie.Intl. Ed. Eng., 32, 666-690; Agrawal et al., (1990). Proc. Natl. Acad.Sci. USA 87, 1401-1405]. The consequence of such interaction is acompetitive blockade of either DNA or RNA synthesis at replication ortranscription, respectively and, in the case of exons, at thetranslational steps in protein synthesis. Upon cell entry, exogenouscomplementary deoxyoligonucleotides hybridize with a target mRNA,inducing an excision endonucleolytic effect on the mRNA, a so-calledribonuclease H(RNAse H) effect resulting in synergism with hybridizationinhibition [Zamecnik et al., (1996) Ed. By Sudhir Agrawal, Humana Press,NJ, 1-11]. Likewise, RNA editing [Simpson et al., (1996) Annu. Rev.Neurosci. 19, 27-52] has been reported in numerous cell systems wherenucleotide sequences can also be modified at the RNA level. RNA editingencompasses various mechanisms, including base substitutions anddeletions [Simpson et al., (1996) Annu. Rev. Neurosci. 19, 27-52]. RNAduplexes with internal and external guide sequences in some cases arerequired to drive these reactions.

Double stranded RNA sequences have a role in RNA interference (RNAi) andgene silencing at the transcriptional level [Hannon, (2002) Nature, 418,244-251]. RNAi mediated gene silencing was discovered in C. elegans[Fire et al., (1998), Nature, 391, 806-811; Montgomery et al., (1998),Proc. Natl. Acad. Sci. USA 95, 15502-15507; Grishok et al., (2000),Science, 287, 2494-2497] and has been observed in numerous cell modelsand organisms [Hannon, (2002) Nature, 418, 244-251; Hammond et al.,(2000), Nature, 404, 293-296; Silva et al., (2002), Trends Mol. Med. 8,505-508]. Gene silencing results from successive cleavage of long dsRNA,particularly originating from viruses, to oligonucleotide siRNAs byDICER enzymes [Zamore et al., (2000) Cell, 101, 25-33; Bernstein et al.,(2001), Nature, 409, 363-366]. After oligonucleotide hybridization ofsiRNA with mRNA, the cleavage of target mRNA is catalyzed [Hammond etal., (2000), Nature, 404, 293-296; Zamore et al., (2000) Cell, 101,25-33]. RNAi phenomena have been used for target gene silencing fromnematodes and plants to mammals [Hannon, (2002) Nature, 418, 244-251].Separate from the above is CpG stimulation of host immunomodulatorymechanisms, in both prokaryotic and eukaryotic organisms [Krieg (2003)Nat. Med., 9, 831-835; Kandimalla et al., (2003) Biochemical SocietyTransactions, 31, 654-658].

The gain of function and/or correction of defective genes present acontinuing challenge for gene therapy. Partial restoration of functionin the Δ508 CFTR background has been shown to occur by membraneinsertion of Δ508 CFTR without changes in either phenotype or genotype[Brown et al., (1996) Cell Stress & Chaperones, 1, 117-125; Arispe etal., (1998) J. Biol. Chem. 273, 5727-3574). This is based on thecapacity of Δ508 CFTR to function even as a misfolded protein [Welsh etal., (1993), Cell, 73, 1251-1254; Ward et al., (1994), J. Biol. Chem.269, 25710-25718]. Its translocation to a plasma membrane mayincompletely restore functionality of the phenotype RNA/DNAoligonucleotide hybrids [Cole-Strauss et al., (1996), Science, 273,1386-1399; Yoon et al., (1996), Proc. Natl. Acad. Sci. USA 93,2071-2076; Alexeev et al., (1998), Nature Biotech. 15, 1343-1346;Parekh-Olmedo et al., (2001), Sci. STKE 73, PL1] and single strandedoligonucleotides [Igoucheva et al., (2001) Gene Therapy, 8, 391-399]have been used for the correction of defective genes. Substituted,circular, single stranded RNA/DNA chimeras have been employed to insertbase pairs in deficient genomic DNA [Cole-Strauss et al., (1996),Science, 273, 1386-1389; Yoon et al., (1996), Proc. Natl. Acad. Sci. USA93, 2071-2076]. Nucleotide exchange of target episomic and genomic DNAis shown using chimeric RNA/DNA oligonucleotides. Some attempts atreplicating this work, however, showed no nucleotide exchange in thetargeted loci by cloning of the PCR products [Zhang et al., (1998)Antisense & Nucleic Acid Drug Developm. 8, 531-536]. Results were viewedby these and other investigators as PCR artifacts created by the RNA/DNAoligonucleotides themselves [Zhang et al., (1998) Antisense & NucleicAcid Drug Developm. 8, 531-536; Taubes, (2002) Science, 298, 2116-2120].Transcriptional repair has been used for repair of mRNA. Spliceosomemediated cis-splicing of pre-mRNA is an essential step in geneexpression [Jurica et al., (2003), Mol. Cell, 12, 5-14]. Othertrans-splicing mechanisms in pre-mRNA molecules have been shown to formfunctional hybrid mRNA molecules in different mammalian systems[Spector, (1993), Curr. Opin. Cell Biol. 5, 442-447; Harris et al.,(1990), Nucleic Acids Res. 18, 3015-3019]. Spliceosome mediated RNAtransplicing (SmaRT) technology has also been used to modify Δ508 CFTRtranscripts in human CF airway epithelia [Liu et al., (2002),Biotechnol. 20, 47-52].

An mRNA hybridized to a single stranded synthetic short piece of DNA wasused to activate RNase H [Agrawal et al., (1990). Proc. Natl. Acad. Sci.USA 87, 1401-1405], which then specifically hydrolyzed only thehybridized segment of mRNA. RNase H as an ubiquitous enzyme had beenpreviously known [[Agrawal et al., (1990). Proc. Natl. Acad. Sci. USA87, 1401-1405]]. The present invention defines nucleotide limits ofRNase H activity in a precise way. At least a 4-6 nucleotide internallylocated region in an mRNA, which is hybridized to a phosphorothioatemodified (PS) oligodeoxyribonucleotide, is needed for Rnase-mediatedexcision. If a precise small segment of mRNA, which included the deletedUUU region (the ribosomal substitute for TTT) were synthesized andannealed to the larger chimera, which induced/activated a small deletionin mRNA, an insertion of the somewhat larger total piece excised mightrestore the phenotypic effect at the mRNA level.

Initially, a modified oligodeoxyribonucleotide with a central segmentcontaining a phosphorothioate (PS) modification was constructed, withflanking segments PS plus 2′-O-methyl modifications. This was designedto hybridize in Watson-Crick base complementarity to the region of Δ508mRNA where the PS section would be directly opposite the Δ508 mRNAposition, flanked 3′- and 5′- by an adjacent few nucleotidescomplementary to the Δ508 mRNA. In this way the PS segment wouldactivate endogenous RNase H, and cleave those bases opposite the PScomplementary oligonucleotide. The 2′-O-methyl PS, plus segments on bothsides of the RNase H sensitive section would be generally nuclease andRNase H resistant and would serve in this way as a “genetic band aid”.Without being limited by any particular mechanism, the flanking segmentsare intended to hold the 5′- and 3′-segments of Δ508 mRNA in place, inposition for a possible insertion or repair of bases in a second step.For the possible repair step two single complementary strandoligonucleotides were constructed: Cystic Fibrosis 4 (CF4) and CysticFibrosis 6 (CF6), as shown in Scheme 1. This combination was annealed toform a duplex. The duplex was added to growing Δ508 CFTR cell cultures.Patch clamp examination [Reisin et al., (1994) J. Biol. Chem. 269,20584-20591] showed evidence of phenotypic reversion (FIGS. 1 and 2).When the CF4/CF6 annealed duplex without oligonucleotide treatment, asdescribed above, was, however, added to the tissue culture cells,phenotypic reversion was likewise found, based on the patch clamptechnique. A one step phenotypic reversion technique was chosen, as asimpler model for the sequencing studies described below.

CF4 and CF6 were annealed to form an oligonucleotide duplex (CF4/CF6)(Table 1) and were then added to the Δ508 cells in tissue culture. Therewas restoration of the CFTR channel to functional normality, asdetermined by patch changing and 6-methoxy-N-(3-sulfopropyl)quinolinium(SPQ) quenching techniques. Under those conditions, restoration offunction was found to occur in 8-18 hours (FIGS. 1 and 2). As2′-O-methyl modified CF4 has been reported not to activate RNase H [Shenet al., (1998) Bioorg. Med. Chem., 6, 1695-1705], there would appear tobe another endonucleolytic enzyme responsible for the base excision,unless RNase H is activated by a triple-stranded oligonucleotide, inwhich the complementary 2′-O-methyl moiety is present.

Under Rose experimental conditions there was complete restoration ofCFTR function, whereas non-insertion reversions have been only partial.[Rose D M et al, Eur J Res 2000; 5, 9-12] On replication of the humantissue culture cells and washing out of the oligonucleotide after a fewdays incubation, the restoration of CFTR function was lost. Thisstrongly indicated a phenotypic but not genotypic (i.e. RNA but not DNA)restoration of functionality of the CFTR channel. Amplification andsequencing technology was used to determine whether trinucleotideinsertion into Δ508 mRNA has been made. Scheme 1 is a schematic diagramof the insertional mechanism.

An oligonucleotide complex (CF4/CF6, respectively 2′-0-methylRNA/unmodified RNA oligonucleotide duplex) was used herein to restoreCFTR function by insertion of missing bases in Δ508CFTR mRNA from acultured (Δ508) cell line. Cyclic AMP-activated (cAMP) whole cellcurrents and Cl⁻ transport were detected in CF4/CF6 treated, rather thancontrol Δ508 cells by the patch clamping and by SPQ fluorescencequenching analysis, respectively. Further, the nucleotide addition inthe deleted region of Δ508 CFTR was determined after amplification bythe Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Insertionof UGU and replacement of U by C immediately 5′ to the deletion site inΔ508 mRNA appear to have taken place, with phenotypic but not genotypicreversion in tissue culture of treated cells.

Genetic Diseases/Disorders

Suitable genetic diseases and sequences of appropriate targets in genes,such as those listed in U.S. Patent Application Publications20030051270, 20030217377 and 20040014057, each incorporated herein byreference in there entireties, are treated using designedoligonucleotide complexes and the methods provided herein.

Several genetic diseases relate to disorders in the sequences of mutatedgenes and genes with inborn errors. Other major sources of geneticdiseases, shown above, are due to “switch on” silent genes or presenceof viral genes in the mammalian genome.

The cause of a fatal or serious genetic disease may be as small as asingle mismatch in base pair (one mismatch, one inserted/deleted basepair in the whole gene sequence) or absence of a partial or whole genein the genome. Consequently, availability of the highly specificoligonucleotide complex technique for the restoration of the targeted(defected) gene could be a powerful tool to combat currently incurablegenetic diseases.

Oligonucleotide complexes and methods for their use for the functionalrestoration of the mutated genes by deletion/insertion mechanism of thesegment of RNA are provided. Site-specific insertion of the genes withthe oligonucleotide complex technique (as shown herein as replacement ofa cystic fibrosis phenotype in a cultured Δ508CFTR cell lineexperiments) can occur with a one or two step oligonucleotide complextreatment. Below are shown schemes for restoration of mutated and inbornerror genes (Schemes 2-7), and schemes of switch-on and/or switch-offtargeted genes (Schemes 8-10) using an oligonucleotide complextechnique.

One step and two step oligonucleotide complex treatment of targeted R NA(or DNA) are shown in the Schemes 3, 5, 7, 9 and 2, 4, 6, 8, 10respectively.

Scheme 2 shows a site-specific deletion/insertion mechanism at thetargeted region of RNA with the two-step oligonucleotide complextechnique. The targeted region in this scheme could be one (or a few)mismatched bases or one (or a few) inserted bases in the RNA sequence.On the first step, oligonucleotide for RNase-H activation deletes thetarget region of RNA, and on the second step insertion of the desirablesequence takes place. The RNase H deleted region could be a few or morebases, depending on the extend of RNase induced deletion, as a result ofwhich the RNA oligonucleotide from the insertion duplex might be splicedinto the deleted region, or alternatively serve as a triplex backbonesequence for a one-by-one insertion mechanism.

Scheme 3 shows a site-specific deletion/insertion mechanism of thetargeted region of RNA with the one step oligonucleotide complextechnique. There are different possibilities of one step restoration:triplex backbone sequence formation by a one-by-one insertion mechanism,with specific docking and cleavage of selective nucleotide sequences,which are hybridized to an RNA (or DNA) sequence. The cleavage might byinduced by either a new enzyme or the Dicer enzyme, and as a result,production of RNA (or DNA) with a corrected (inserted) region.

Scheme 4 is shown site-specific insertion mechanism of the deletedregion of RNA with the two-step oligonucleotide complex technique.Schemes 6, 8 and 10 show a site-specific insertion/deletion mechanism atthe targeted regions of RNA (or DNA) with the two-step oligonucleotidecomplex technique. RNA (such as Huntington's disease) in Scheme 6contains reiteration of excess triplets, and accordingly the purpose ofthe deletion step is to cut out the excess repeats. RNA shown in Scheme8 is a model of active (harmful) RNAs and the goal of the insertion isto switch off these RNAs. The purpose of the insertion shown in Scheme10 (opposite of Scheme 8) is to “wake up” a silent gene or to create newone. The insertion shown in Schemes 2, 4, 6, 8 and 10 could proceed withsimilar mechanisms, but goals achieved for each of the approach would bedifferent.

The purpose of the approaches shown in Schemes 5, 7 and 9 is identicalto the Schemes 4, 6 and 8. The deletion/insertion mechanism of thetargeted region of RNA, shown on Schemes 5, 7 and 9, proceeds with theone step oligonucleotide complex technique, as described for Scheme 3.

Schemes 11 and 12 show schemes for repair of an inactive tyrosinase geneby one and two step oligonucleotide complex approaches, respectively.The schemes contain one of the possible versions of the oligonucleotidecomplex which may be used for the replacement of mutated C with G ininactive tyrosinase mRNA. This type of alteration can achieve aphenotypic restoration of the mutated tyrosinase gene.

Schemes 13 and 14 show schemes for repair of Cystic Fibrosis Δ508 mRNAby one and two step oligonucleotide complex treatment approach. Asdescribed herein, mutated Δ508 mRNA contains two defects: first, absenceof a UUU triplet at Δ508 region and change of U to a C immediately 5′ tothe same Δ508 position. Because of the close location of both mutationsone set of oligonucleotide complex was sufficient for repair of bothdefects.

In some cases the targeted gene might contain more then one mismatch orinserted (deleted) region, which may be distantly located. Forrestoration of such genes it would be appropriate to use two or moreoligonucleotide complex sets consecutively or simultaneously.

For enhancing targeted gene repair efficiency of oligonucleotidecomplex, the complex could be used synergistically with other known orpotential therapeutic compounds.

This application claims priority to U.S. Ser. No. 60/557,732, filed onMar. 29, 2004 and U.S. Ser. No. 60/560,026, filed on Apr. 7, 2004, eachof which is incorporated herein by reference in their entireties.Further, the contents of all references cited are incorporated in theirentireties herein by reference. Practice of the invention will be morefully understood from the following examples, which are presented hereinfor illustration only and should not be considered as limiting theinvention in any way.

EXAMPLES

The examples provided herein demonstrate feasibility of using anoligonucleotide complex technique as a therapeutic and/or prophylacticagent for the phenotypic and restoration of function of a mutated gene.The following Material and Methods were used throughout the Examples.

Oligonucleotide Synthesis

DNA and RNA oligonucleotides (Table 1) were synthesized on a 394 DNA/RNAsynthesizer (Applied Biosystems) with phosphoramidite chemistry andstandard phosphoramidite monomers from Glen Research. For theintroduction of 3′ and 5′ phosphate groups on the CF4 oligonucleotide(Table 1), 5′- and 3′-phosphorylation reagents were used accordingly(Glen Research). Phosphorothioate bonds were introduced by sulfurizationwith Beaucage thiolating reagent [Padmapriya et al., (1994) AntisenseRes. Dev. 4, 185-199]. 2′-0-methyl modifications, oligoribonucleotidesand phosphorothioate oligonucleotides were synthesized, and HPLCpurified as described [Metelev et al., (1994) Bioorg. Med. Chem. Lett.4, 2929-2934; Agrawal et al., (1998) Antisense Nucleic Acid Drug Dev. 8,135-139].

Annealing of CF4/CF6 Oligonucleotide Duplex

Stock solutions of CF4 (1 mM) and CF6 (1 mM) were prepared by dissolvingcompounds in high ionic strength buffer (0.2 M NaCl, 20 mM MgCl₂, 20 mMTris-HCl, pH 7.0). Duplex formation was prepared with 1:1 (v:v) mixtureof compounds, by heating to 75-80° C., and cooling down gradually toroom temperature. All duplexes and compounds were sterilized by passagethrough 0.45 μm cellulose acetate centrifuge filters (Costar).

Cell Culture and Incubation

Mouse mammary carcinoma cells (c127i) transfected with human epithelialCFTR wild type (WT1) or Δ508 CFTR cells [Cantiello et al., (1994), J.Biol. Chem. 269, 11224-11232; Dechecchi et al., (1993) J. Biol. Chem.268, 11321-11325] were used for these studies. The transfected c127icells are high level producers of Δ508 CFTR mRNA. Cells were grown andmaintained in Dulbecco's medium (DMEM), supplemented with 10% fetalbovine serum and 1% L-glutamine, as previously reported [Verkman, (1990)Am. J. Physiol. 259, C375-C388].

Electrophysiology

Whole-cell and excised inside-out patches were obtained to assesscAMP-PKA dependent anion currents in treated Δ508 cells. Currents andcommand voltages were obtained and driven. respectively, with a Dagan3900 amplifier using a 1 gigaohm headstage. The excised patch-clampconfiguration was carried out as previously described [Reisin et al.,(1994) J. Biol. Chem. 269, 20584-20591]. Single channel data wereobtained between ±100 mV in symmetrical Cl⁻. Data were further analyzedas previously described [Reisin et al., (1994) J. Biol. Chem. 269,20584-20591]. The pipette and bathing solution contained, in mmol/L,either: NaCl 140, MgCl₂ 1.0, KCl 5, and N-2-hydroxyethylpiperazineN′-2-ethanesulfonic acid (HEPES) 10, at pH 7.4, or MgCl₂ 70, HEPES 10,pH 7.4. The bathing solution also contained 1.0 mmol/L CaCl₂. Wheneverindicated, the patch-pipette was filled up to at least one third of itsheight with either MgATP or TrisATP (100 mmol/L, pH 7.4 adjusted withN-methyl-glucamine) as previously reported [Reisin et al., (1994) J.Biol. Chem. 269, 20584-20591]. Experiments were conducted at roomtemperature. The cAMP stimulatory mixture contained 8-Br-cAMP,isobutyl-methyl-xanthine (IBMX) and forskolin. Final concentrations were500 μmol/L, 200 mol/L, and 10 μmol/L, respectively. The catalyticsubunit of PKA was used at a final concentration of 20 μg/ml. The Cl⁻channel blocker, diphenylamine-2-carboxylate (DPC) was kept in a100-fold stock solution (20 mmol/L) in 50% water/ethanol. DPC was usedat a final concentration of 500 μM.

SPO Fluorescence

Cyclic-AMP-stimulated Cl⁻ transport was also followed by fluorescencechanges of cells loaded with the Cl⁻ sensitive dye6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) [Verkman, (1990) Am. J.Physiol. 259, C375-C388]. Briefly, cells were grown to partialconfluence on glass coverslips. SPQ cell loading was conducted bya15-min incubation in a Cl⁻ free diluted salt solution containing SPQ (5mM). The saline solution contained 135 mM Na⁺-gluconate, 2.0 mM KH₂PO₄,1.0 mM MgSO₄, 1.0 mM Ca²⁺-gluconate, and 10 mM HEPES (pH 7.4). In someexperiments, gluconate was replaced by either isethionate or aspartatewith similar results. SPQ fluorescence was determined under an oilimmersion X20 objective in a Nikon E-800 fluorescence microscope (Tokyo,Japan), with an UV filter (96101C UV-2E/C). Images were captured with aHamamatsu (C4742-95) digital camera and stored as TIFF files in aMacintosh computer with the IPLab Spectrum software (Signal AnalysisCorp.). Pictures were analyzed digitally with the NIH Image 1.62b7software. SPQ cytoplasmic fluorescence values were normalized betweenintra-nuclear (considered as zero quenching, i.e. low Cl⁻), andextracellular background. Data were expressed as percent fluorescencewith respect to time zero, the time when cells were placed in anisotonic saline solution. The solution contained 135 mM NaCl, 2.0 mMKH₂PO₄, 1.0 mM MgSO₄, 1.0 mM Ca²⁺-gluconate, 10 mM HEPES (pH 7.4), withor without the cAMP stimulatory combination mixture.

Primers and RT-PCR of Δ508CFTR mRNA

Total RNA isolated by Trizol® reagent according to the manufacturer'sprotocol (Invitrogen, CA) from either WT1 or Δ508 cells treated with orwithout CF6/CF4 annealed duplex oligonucleotides (final concentration ofduplex in reaction mixture 101) was used to perform the RT-PCR assay[Kleppe et al., (1971) J. Mol. Biol. 56, 341-361] in two steps by usinga ThermoScript RT-PCR system (GIBCO, BRL). Several sets of primers wereused (see CF Table 1). In the first step, total RNA (˜2 μg) wasincubated for 60 min at 55° C. with amplification refractory mutationsystem (ARMS) [Ferrie et al., (1992) Am. J. Hum. Genet. 51, 251-262]reverse mutant (M1) and normal (N1) primers (0.5 μg each) separately forthe first-strand synthesis. In a second step, after heating at 94° C.for 5 min, 35 cycles of PCR were carried out on the samples. PCR cycleswere performed first by denaturation at 94° C. for 2 min, annealing at62° C. for 1 min, and extension at 72° C. for 2 min, followed by a finalextension for 10 min at 72° C. Two ARMS reverse primers (N1 and M1)[Ferrie et al., (1992) Am. J. Hum. Genet. 51, 251-262], and a forward(F1) primer were used (Table 1). Similarly RT-PCR was performed withARMS PS reverse primer (SCFR) and PS forward primer (SNF1, Table 1).RT-PCR products were separated in 3% agarose gel and subjected toautomated DNA sequence analysis.

Similarly, RT-PCR was conducted using allele specific primers (Scheme1B, Table 1) as follows. In the first step, total RNA (˜2 μg) wasincubated for 60 min at 55° C. with reverse (CFR) primer (0.5 μg each)for the first-strand synthesis. In a second step, samples were heated at94° C. for 1 min. PCR was performed for thirty cycles at 60° C.including denaturation at 94° C. (45 sec), annealing at 60° C. (45 sec),and extension at 72° C. for 1 min. This procedure was ended with aseven-minute final extension at 72° C. Two forward primers (CFW and CFM)and one reverse primer (CFR) were used for allele specific RT-PCR (Table1). The RT-PCR assay was performed with the MasterAmp™RT-PCR system(Epicentre, WI). In this procedure, combined reverse transcription andPCR were performed in the presence of forward normal (NF1) and mutant(MF2) and reverse (CFR) primers (0.5 μg each) separately. First, sampleswere incubated for 20 min at 60° C. PCR was performed for 40 cycles at94° C., denaturation for 30 sec, annealing at 62° C. for 30 sec and 72°C. extension for 1 min. The reaction ended by a final extension for 6min at 72° C. Two forward primers (NF1 and MF2) and a reverse primer(CFR) were used for this procedure. RT-PCR products were separated in a3% agarose gel and were then subjected to automated DNA sequenceanalysis. ThermoScript RT-PCR products obtained by ARMS primers weresubcloned for further separation and purification of possibleheterogeneity of oligomers so obtained in pCR-Blunt vector, according tothe manufacturer's protocol (Invitrogen, CA). The resulting clones weresubjected to automated DNA sequence analysis using a T7 promoter primer.

Example 1 Restoration of Δ508CFTR Phenotype

To restore the normal (wild type) phenotype in cells expressingΔ508CFTR, modified oligonucleotides were constructed and their effect onion transport was assessed in Δ508 cells treated with these constructs.In a first step, a modified oligodeoxyribonucleotide was synthesized,containing phosphorothioate (PS) and PS plus 2′-O-methyl modificationsfor the central and flanking segments, respectively. Thisoligonucleotide was designed to hybridize in Watson-Crick basecomplementarity to the region of Δ508 mRNA where the PS section would bedirectly opposite the Δ508 mRNA position, flanked 3′- and 5′- byadjacent nucleotides complementary to the Δ508 mRNA. Accordingly, the PSsegment would activate endogenous RNase H, and cleave those basesopposite the PS complementary oligonucleotide (10 μM). The insertion ofbases then occurs in a second step.

For the second or insertion step, two complementary single strandoligonucleotides, CF4 and CF6 (Table 1, Scheme 1A) were constructed. Theannealed CF4/CF6 duplex (10 μM final concentration) was added to theΔ508 CFTR cells after 2 hours incubation, then washout, in the deletionstep. Next, following overnight or up to 72 hour incubation in thesecond (insertion) step, patch clamp examination was conducted on thetreated cells.

Whole cell currents of two step treated Δ508 cells (FIG. 1) showed a243% increase after cAMP stimulation (2.98±0.68 nS/Cell vs. 0.87±0.16nS/Cell, n=24, p<0.01), which was absent in the control Δ508 cells (FIG.1). The cAMP-activated currents were largely (>84%) inhibited by theCFTR inhibitor DPC (500 μM) as expected for wild type CFTR. Similarresults were obtained with WT1 cells, over-expressing wild type CFTR inthe same cellular background. The treated Δ508 cells (10/12) displayedPKA and ATP activated 10-12 pS Cl⁻ channels (FIG. 2) not observed in thecontrol Δ508 cells (0/24).

When the CF4/CF6 annealed duplex was, however, added to the tissueculture cells without performing a first oligonucleotide treatment stepas described above, phenotypic reversion was likewise found, asdetermined by the patch clamp technique. Thus, a one step phenotypicreversion technique was chosen as a simpler model for the sequencingstudies described below.

Surprisingly, CF4/CF6 cells sporadically showed large, DPC inhibitablewhole-cell currents in the absence of cAMP simulation (data not shown).Both WT1 and CF4/CF6 treated Δ508 cells responded with a comparablysimilar change in SPQ fluorescence in response to cAMP stimulation (FIG.3) [Ram et al., (1989) Proc. Natl. Acad. Sci. USA 86, 10166-10170].Control Δ508 cells showed no cAMP-induced change in SPQ fluorescence(FIG. 3). The data indicate that CF4/CF6 treatment of Δ508 cellsrestores a normal tissue culture phenotype, consistent with the presenceof functional CFTR.

Example 2 Sequencing Analysis of PCR Products

In order to determine the extent of restored phenotype in the CF4/CF6treated Δ508 cells, total RNA was isolated and CFTR specific primerswere used to amplify the predicted region. CFTR wild type specificprimers efficiently amplified single bands of the expected size in theCF4/CF6 oligonucleotide treated cells. The sequence region after thistreatment did not show changes in the PCR amplified oligonucleotideswhen wild type primers were used. Because the number of Δ508 mRNA copiesthat are potentially repaired may be low, allele specific primers foreither wild type or mutated CFTR were used next (CFFW and CFFM, Table1).

Allele specific primers detected mRNA from serial dilutions of wild andΔ508 total RNA by means of a shift of the amplified band (FIG. 4). Wildtype RT-PCR product could be detected in 1:10,000 dilutions in Δ508 mRNAbackground (FIG. 4). Initially, RT-PCR product from the allele specificwild type primer (CFFW, Table 1) in the oligonucleotide treated Δ508cells also failed to detect insertion in the amplified band. Therefore,total RNA of CF4/CF6 treated Δ508 cells were further tested by PCRanalysis with ARMS specific primers (FIG. 5) [Skerra, (1992) NucleicAcid Res. 20, 3551-3554]. Wild type and mutated ARMS primer-amplifiedPCR bands were examined by DNA sequence analysis. The sequence of theamplified mutated cDNA showed a variety of one-codon insertions, rich inG residues. Whether a GGG codon (glycine) is an acceptable substitutefor phenylalanine remains to be determined, since systematic study ofsuch base insertion has not been done to our knowledge.

To analyze this observation further, the PCR products were subcloned inpCR-Blunt vector and subjected to DNA sequence analysis. The fraction ofcontrol RNAs isolated from untreated Δ508 cells showed nooligonucleotide insertion. However, mRNA isolated from CF4/CF6 treatedΔ508 cells showed 20-30% UGU insertion, based on analysis of thepercentage of subclones showing TGT insertion into the RT-PCR generatedΔ508 DNA. This percentage of insertion is apparently sufficient forphenotypic reversion in the tissue culture system. None of the subcloneduntreated Δ508 cells displayed false positives (three-base insertion) inthe region flanking the initial deletion (FIG. 6). Combined reversetranscription and PCR were performed with forward wild type (NF1) andmutant (MF2), and reverse (CFR) primers (0.5 μg each) separately.Insertion of bases in the proper position (Scheme 1B) has been found insubcloned and sequenced RT-PCR products, from treated but not controlΔ508 cells.

Without being limited by any particular mechanism, the hypothesis wastested as to whether base insertion in the treated Δ508 cells hasoccurred at the RNA level. It was possible that the repair mechanism wascarried out instead of/or in addition to, the DNA level, by way ofreverse transcription extending back to the genome. This was tested byRT-PCR analysis of DNA from subcloned Δ508 cells originally treated withCF4/CF6. No evidence was obtained that the restored phenotype wascarried back to the inheritable DNA genome level was obtained. Westernblot analysis was conducted with antibodies targeted to epitopesupstream and downstream of the Δ508 deletion, respectively. The dataindicate that the full-length protein was made in the presence of theoligonucleotides.

Further, the data rule out that partially degraded protein was beingtranslated after treatment with the oligonucleotides because of stopcodon missing signals in the treated mRNA [Bedwell et al., (1997) NatureMed., 3, 1280-1284]. Both antibodies showed the same level of protein,without shorter, truncated or degraded peptides. Thus, treatment withCF4/CF6 does not act as an inhibitor of protein synthesis.

Example 3 Mechanism of Insertion

As an initial step, restoration was sought of the most common CFphenotype by antisense oligodeoxynucleotide hybridization to the regionimmediately adjacent to the trinucleotide deletion, on both sides of theΔ508CFTR mRNA. The double-stranded synthetic2′-O-methyl-RNA/unmodified-RNA oligonucleotide chimera (CF4/CF6) wasconstructed and used to anneal, selectively cut, and repair the missingregion (Scheme 1B). This duplex, as shown, contains a single stranded2′-O-methyl-substituted 33-mer oligoribonucleotide (CF4) hybridized toan unmodified 11-mer oligoribonucleotide with 5′ and 3′ monophosphatetermini (CF6). The role of the 5′- and 3′-phosphate termini of CF6, ifany, remains to be determined. The CF4/CF6 chimera may theoreticallyhybridize with the mRNA bearing the deletion (Scheme 1B), followed by anmRNA cleavage step, as a result of which CF6 might theoretically bespliced into this region or alternatively, serve as a triplex backbonesequence for a one-by-one insertion mechanism.

Without being limited by any particular mechanism, the mismatch createdby using the oligonucleotides provided herein and found immediatelyproximal in the 5′-direction to the Δ508 mRNA UUU deletion may induce anas yet unknown deletion mechanism. For one, the deletion step may beinduced by either a new enzyme or by the Dicer enzyme. Anotherpossibility is that RNAse H, in the presence of a triplex, may inducethe deletion. There may conceivably be a deletion in RNA analogous tothe MutS DNA mismatch mechanism [Wang et al., (2003) Proc. Natl. Acad.Sci. USA 100, 14822-14827]. The process by which CF4/CF6 restores thephenotype is consistent with specific docking and cleavage of selectivenucleotide sequences hybridized to an mRNA sequence. A -TTT- insertioninto the PCR amplified deoxyoligonucleotide would be expected, if the-UUU- from CF6 had been inserted into the Δ508 mRNA. However, TGT (UGUin the Δ508 mRNA) insertion was consistently observed in clones obtainedby ARMS primers amplification RT-PCR products (Scheme 1B and FIG. 6).This finding suggests a one-by-one insertion mechanism, with a G inplace of a U.

Initial sequencing of RT-PCR oligodeoxynucleotides obtained with ARMSprimers specific for the CFTR wild type sequence (N1, Table 1) revealedthe presence of a mixture of residues rich in G's in the Δ508 site.Subcloning of this oligodeoxynucleotide band resulted in the findingthat some but not all of the sequenced cDNA material expressed a TGT atthis Δ508 site. Since it was mRNA that was subjected to RT-PCRamplification, the corresponding bases in the Δ508 region were actuallyUGU. The G residue in the UGU could be accounted for by a restorationmechanism with other than Watson-Crick complementarity in insertion[Yang et al., (2003) Proc. Natl. Acad. Sci. USA 100, 15376-15380]. The3′ hybridization initial steps enabled by the ARMS primers may also beflawed by exonucleotide-induced primer degradation in the reversetranscriptase step of the RT-PCR amplification procedure [Skerra, (1992)Nucleic Acid Res. 20, 3551-3554; Smith et al., (2003) Proc. Natl. Acad.Sci. USA 100, 15440-15445]. Other possibilities of error introduction[Kobayashi et al., (1990) Am. J. Hum. Genet. 47, 611-615], may explainthe apparently artifactual deletions shown in Scheme 1B which accompanyinsertions. This consideration was partially confirmed by wild type ARMSprimer-RT-PCR product amplification of the sequence in WT1 cells, whichonly carries wild type CFTR. In this reaction, the expected -TTT- wasfound by sequence analysis of the wild type RT-PCR material, in contrastto the TGT (actually UGU in mRNA) when the repaired Δ508 mRNA wassequenced.

For further clarification of the above results, new sets of primers wereconstructed with an initial phosphorothioate substituted nucleotide inthe 3′ end, followed by several (PO) standard nucleotides. This improvedprimer selectively amplified CF4/CF6 treated, but not control(untreated) RT-PCR product in Δ508 cells. The ARMS forward PS primer(SNF1) inserts UGU without a concomitant new deletion (Scheme 1B, Table1), thus presenting the best case for phenotypic reversion to wild type.Just 5′ to the UUU in the wild type CFTR is a C residue, while in themutant Δ508 CFTR this residue is a U. This may conceivably result inreplacement failure 5′ to the inserted UGU using the ARMS reverse primer(N1). The purpose in using CF4/CF6 complex, which has a G residue ratherthan U proximal to the 5′ end of the Δ508 deletion, was to make thisresidue complementary to that in the wild type sequence rather than inthe deleted Δ508 sequence. Both AUC and AUU code for isoleucine. Thisbase mismatch for the Δ508 mRNA (CF Scheme 1) may however induce asingle strand break, analogous to that found for single DNA mismatchesas mentioned above [Wang et al., (2003) Proc. Natl. Acad. Sci. USA 100,14822-14827], necessary for a subsequent repair mechanism to beinitiated. This eliminates the possibility that the change of C to a U,immediately 5′ to the TTT in the same position in the Δ508 gene maycontribute, in addition to the Δ508 TTT deletion, to the phenotypicchange in CFTR.

DNA polymerase has a high degree of Watson-Crick fidelity insynthesizing complementary strands. Reverse transcriptase, which startsat the 3′-end of the PCR amplification, however, has a lower level ofthis specific type of fidelity. This property of reverse transcriptasemay be a possible explanation for the UGU (the equivalent of TGT) foundin the amplified, restored Δ508 mRNA. In relation to phenotypicrestoration, benign mutations of the TTT present in the wild type genedo exist. TGT is one of these, coding for cysteine, which appears to bean acceptable substitute for phenylalanine in the Δ508 region [Kobayashiet al., (1990) Am. J. Hum. Genet. 47, 611-615]. The 2′-O-methyl group,plus the 5-methyl of thymidine, which uridine does not have, may alsoalter the tertiary structure of the CF4 chimera. Such factors mayinfluence the nucleophilicity, electrophilicity, and polarizability ofthe bases, which make up the mRNA-CF4/CF6 triple-stranded structure.Tinoco and colleagues have described numerous double stranded DNA/RNAbase complementarities which conformational and other experimentalmolecular conditions may cause to favor over the standard Watson-CrickAT and GC ones [Burkard et al., (1999) In The RNA World. editors. ColdSpring Harbor Lab. Press, New York. 675-680]. The highly sensitivenanosphere/gold procedure of Letsinger and colleagues [Taton et al.,(2000) Science, 289, 1757-1759; Letsinger et al., (2000) Bioconj. Chem.1, 289-291] may in the future be applicable as an alternative to the PCRtechnique, avoiding possible artifacts introduced by PCR amplification,or else may be used following subcloning.

The data herein indicate that specific base insertion in Δ508 mRNA hasbeen made. Certain PCR-introduced artifacts have been avoided byphosphorothioate modification of the 3′-terminal residues of ARMSspecific primers. Under our best conditions thus far, insertion of UGUhas taken place in a sufficient fraction of Δ508 mRNA to inducephenotypic but not genotypic reversion in a tissue culture cell line.The subcloning data showed 20-30 percent insertion of UGU in the site ofthe Δ508 mRNA UUU triplet deletion.

Example 4 Regulation of Huntingtin Protein Gene and OculocutaneousAlbinism Aim-1 Gene

The methods of the instant invention have also been applied to thegenetic disorders Huntington's disease and Albinism. Specifically, thegene deletion/insertion mechanism has been applied to the Huntingtinprotein gene [Cell 72: 971-983, 1993] and the Oculocutaneous AlbinismAim-1 gene [J Biol Chem. 2002 Jan. 4; 277(1):402-6].

The gene deletion/insertion for the Huntingtin gene inhibits translationof Huntingtin protein, which synthesizes too many consecutive glutamineresidues. Oligonucleotide compounds have been prepared for thisinhibitory purpose (Table 2). Deletion of two codons of mRNA (+10+15from translation site) and insertion stop codons (UAA) can inhibittranslation of Huntingtin protein. Table 2 shows the HT3 and HT4/HT6oligonucleotides and Scheme 15 shows the insertion into the gene forHuntingtin protein with HT3 followed by HT4/HT6 duplex oligonucleotides.Scheme 15 can presumably be carried out in a one step process using theHT4/HT6 oligonucleotide complex.

Absence of 7 bases in Aim-1 gene is the cause of Oculocutaneousalbinism. The gene deletion/insertion mechanism can be utilized for theAim-1 gene as shown in Scheme 16. Oligonucleotide compounds have beenprepared for this purpose (Table 2). Table 2 shows the Alb3 andAlb4/Alb6 oligonucleotides and Scheme 16 shows the insertion into theAim-1 gene with Alb3 followed by Alb4/Alb6 duplex oligonucleotides.Scheme 16 can presumably be carried out in a one step process using theAlb4/Alb6 oligonucleotide complex.

TABLE 1 Sequences of oligonucleotides used (5′ > 3′)

Primers and constructs used to repair CFTR mRNA. Lower case letter are anucleotide sequence in CF4 corresponding to bases of 2′-O-methylribosyloligonucleotides, with internucleoside phosphate bonds. Upper caseletter are a nucleotide sequence in CF6 corresponds to natural RNAsequences and small case p corresponds to the terminal phosphate groups.Bold type corresponds to the natural DNA sequences. Shaded region inSCFR, SNF1 and SMF2 corresponds to internucleoside phosphorothioatebonds.

TABLE 2 Sequences of oligonucleotides used (5′ > 3′)

Primers and constructs used to repair Huntingtin protein gene and theOculocutaneous Albinism Aim-1 gene

OTHER EMBODIMENTS

Although particular embodiments have been disclosed herein in detail,this has been done by way of example for purposes of illustration only,and is not intended to be limiting with respect to the scope of theappended claims, which follow. In particular, it is contemplated by theinventors that various substitutions, alterations, and modifications maybe made to the invention without departing from the spirit and scope ofthe invention as defined by the claims. Other aspects, advantages, andmodifications are considered to be within the scope of the followingclaims. The claims presented are representative of the inventionsdisclosed herein. Other, unclaimed inventions are also contemplated.Applicants reserve the right to pursue such inventions in later claims.

1. An RNA oligonucleotide complex for modulating the expression oractivity of a cystic fibrosis transmembrane conductance regulator (CFTR)gene product, the complex comprising a first oligonucleotide and asecond oligonucleotide, said first oligonucleotide comprising thenucleic acid sequence of auc aua gga aac acc aaa gau gau auu uuc uuu(SEQ ID NO: 1) and said second oligonucleotide comprising the nucleicacid sequence of pC AUC UUU GGU Gp (SEQ ID NO: 2), wherein said firstand second oligonucleotide are annealed.